Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1

Even slight imbalance between the growth rate of different organs can accumulate to a large deviation from their appropriate size during development. Here, we use live imaging of the pharynx of C. elegans to ask if and how organ size scaling nevertheless remains uniform among individuals. Growth trajectories of hundreds of individuals reveal that pharynxes grow by a near constant volume per larval stage that is independent of their initial size, such that undersized pharynxes catch-up in size during development. Tissue-specific depletion of RAGA-1, an activator of mTOR and growth, shows that maintaining correct pharynx-to-body size proportions involves a bi-directional coupling between pharynx size and body growth. In simulations, this coupling cannot be explained by limitation of food uptake alone, and genetic experiments reveal an involvement of the mechanotransducing transcriptional co-regulator yap-1. Our data suggests that mechanotransduction coordinates pharynx growth with other tissues, ensuring body plan uniformity among individuals.

these perturbations.A minimal model is used to demonstrate that body volume is ultrasensitively coupled to the pharynx size.Finally, the authors show that inhibition of yap-1 (Yes-associated protein/YAP) via RNAi results in impaired larval development -but only when the growth of the epidermis is inhibited.I believe this perhaps one of the first papers to measure scaling in a multicellular organisms by making careful measurements of organ and body size.
Here are some comments that need to be addressed before publication: I am uncomfortable with the generalisation that pharynx is the same as all organs.While the authors are careful in some places, I think it would be more prudent not to generalise the results to all organs (including in the title).There are many reasons to believe other organs may not be similarly linked to body size.
One important comment regarding the organisation and the argument of the paper regarding ultra sensitivity is the way the model is presented.By looking at the data (fig 4b), that is fit to the Hill functon(with a Hill coefficient of 6!), isn't it obvious that f(dp) cannot be 1, or 1-dp?The authors state in line 243 that dp is defined by a hill function.The model is laid out further down in the paper, and other functional forms of dp are tested.I find the argument a bit circular.Some reorganisation or further justification is required.
There is lot of literature that discusses size scaling, and I was surprised to see that the authors have not really touched on that topic, not even in the introduction.This might make a nice addition to motivate the paper.

Some minor comments:
Wherever applicable can authors use volume instead of size?Line 91 -When defining µ_abs -is this (dV/dt)/V) -can it be defined in those terms.
Line 116 "We distinguished three different scenarios" -this referring to previous work -I have seen this work and they define it as adder, size, and folder -it is not labelled the same way in this work.
Line 122 -There's a huge jump going from description of simulations which point to an adder mechanism for the pharynx, to talking about the coupling between the organ and the whole body.How does the simulation account for this coupling?Some explanation is lacking here.
Line 182 -lines are referred as epidermal and hypodermal interchangeably in the rest of the paper -please rectify for consistency.
Line 193 -Please mention the actual value of the slope (m) -this would give an idea of the actual scaling Line 201 -I did not understand the basis for this statement -how do you know the length extension is independent of biomass?Line 215 -"We note that while pharynx-to-body proportions were retained (Fig. 3d, e), pharyngeal depletion of RAGA-1 reduced the overall size (Fig. 3c), indicating that total body size and pharynx-to-body proportions are controlled by separable mechanisms."Again, the basis of this statement is not clear from the previous section.Please clarify the writing.Line 303 -" In conclusion, yap-1 is dispensable for growth and development of C. elegans per se, but its activity is crucial for the robustness of pharynx-to-body size proportions under growth imbalance between tissues."Many assumptions are being made here.Does one know that yap-1 doesn't have some sort of pleotropic effect in later larval stages?Comments on figures:

Role of yap-1
There are some caveats to the analysis of yap-1 and potentially other models for yap-1's role.
a.The role of yap-1 is depicted as a major point in the manuscript based on the prominence in the title and abstract.However, the evidence for the role of yap-1 is solely based on RNAi, which may not be specific.It is important to validate this result with an outcrossed yap-1 mutant.Alternatively, the authors can be explicit about the caveats and reduce the prominence of yap-1 in the manuscript.b.To justify the title that yap-1 mediates ultra-sensitive coupling would require additional experiments to show that the Hill co-efficient is reduced when yap-1 activity is disrupted.
Alternatively, this point can be addressed by revising the title to downplay this specific claim, which would in no way reduce the importance and interest of this work.c. yap-1 signalling is presented as a general mechanism for robustness of proportionate growth.Is it not just as likely that yap-1 specific to the raga-1 pathway as there is literature relating mTOR signalling to Hippo/YAP (e.g., Csibi & Blenis, Nature Cell Biology 2012)?These alternative models should be mentioned in the discussion.

Minor issues:
Does raga-1 affect RNAi efficacy?The authors suggest that yap-1 only comes into play when there is raga-1 perturbation.But there is no evidence provided to rule out the possibility that the RNAi is more severe under raga-1 loss.The point that other RNAi treatments do not synergize with raga-1 does not address this issue since those RNAi treatments may be true negatives.This caveat should be stated in the results section.
Introducing sizing mechanisms (e.g., adder, folder) in greater detail would provide context to make it easier for readers to understand and appreciate many aspects of this work.This work investigates the coupling between pharynx and body growth in C.elegans.Using previously established high throughput fluorescence imaging, and image processing, the authors quantify pharynx and body length (and volume) and find a nice scaling across developmental stages.The authors use an auxin inducible system to try to autonomously perturb pharynx size and hypodermal size.They observe that a similar scaling holds under these perturbations.A minimal model is used to demonstrate that body volume is ultrasensitively coupled to the pharynx size.Finally, the authors show that inhibition of yap-1 (Yes-associated protein/YAP) via RNAi results in impaired larval development -but only when the growth of the epidermis is inhibited.I believe this perhaps one of the first papers to measure scaling in a multicellular organisms by making careful measurements of organ and body size.
We thank the reviewer for endorsing the novelty of our work.
Here are some comments that need to be addressed before publication: I am uncomfortable with the generalisation that pharynx is the same as all organs.While the authors are careful in some places, I think it would be more prudent not to generalise the results to all organs (including in the title).There are many reasons to believe other organs may not be similarly linked to body size.
We agree with the reviewer and have revised the text accordingly, including in the title, which now reads: Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1 One important comment regarding the organisation and the argument of the paper regarding ultra sensitivity is the way the model is presented.By looking at the data (fig 4b), that is fit to the Hill functon (with a Hill coefficient of 6!), isn't it obvious that f(dp) cannot be 1, or 1-dp?The authors state in line 243 that dp is defined by a hill function.The model is laid out further down in the paper, and other functional forms of dp are tested.I find the argument a bit circular.Some reorganisation or further justification is required.
We are thankful for this comment, which allowed to better explain the purpose of comparing different forms of f(dp).The purpose of simulating alternative models is indeed not to validate that f(dp) is a Hill function.Instead, we do this to evaluate the consequence of a deviation from said Hill function on pharynx-to-body length proportions.We believe this comparison is of interest to the reader, as it provides support that maintenance of pharynx-to-body proportions is not a "passive" consequence of food uptake restriction due to a smaller pharynx.We explain this reasoning in the revised text (ln 282ff.), which now reads:

Models that lack of ultra-sensi ve coupling produce large devia ons in pharynx length
Fig. 4a shows that the rela on between body growth and pharynx size follows a steep Hill func on.
We next asked how important is this ultra-sensi ve coupling was for retaining correct pharynx-to-body length propor ons by inves ga ng models with alterna ve expressions for   and   .Specifically, we simulated independent growth of pharynx and body (  1), which would be expected in the absence of any coupling.Second, we modelled propor onal scaling of body growth to pharynx length (  1  ), which could, e.g., occur due to a propor onal limita on of food uptake by a smaller pharynx.Both alterna ve models strongly deviated from the experimental observa ons (Fig. 4c, d) and were insufficient to explain the experimental observa ons.Together, these analyses suggest that propor onal coupling of pharynx and body growth through a reduced food uptake by a smaller pharynx would be insufficient to ensure correct pharynx-to-body length propor ons.
There is lot of literature that discusses size scaling, and I was surprised to see that the authors have not really touched on that topic, not even in the introduction.This might make a nice addition to motivate the paper.
We agree and now motivate our study by the existing literature on size scaling in the introduction, which now reads (ln.48ff): At the scale of organs, size propor ons follow robust allometric rela ons 1,2 , and propor ons of body parts usually scale appropriately over wide range of body sizes 3 .Combined experimental and theore cal work provides elegant explana ons for how ssue pa erning appropriately scales with ssue size during development 4,5 .For example, a nega ve feedback between two diffusible components can ensure scale invariance of morphogen gradients [3][4][5][6] .Gene c experiments revealed ssue autonomous and systemic mechanisms that control organ size 7 .For instance, morphogen gradients are thought to limit the lateral expansion of imaginal discs in Drosophila melanogaster 8,9 and damaged imaginal discs trigger systemic responses via secre on of the relaxin-like signalling pep de Dilp8 10,11 .Similarly, unilateral inhibi on of limb growth in mice triggers a growth response that retains proper limb symmetry 12,13 .However, individual organ growth trajectories have rarely been measured in vivo over me, and how devia ons in organ size are dynamically corrected during development remains poorly understood.

Some minor comments:
Wherever applicable can authors use volume instead of size?
We now specify throughout the manuscript whether we talk about volume or length.In some instances, we refer to a both, length and volume.In these cases, we use the term size to avoid repeated use of lengthy wording of "volume and length".
Line 91 -When defining µ_abs -is this (dV/dt)/V) -can it be defined in those terms.Line 116 "We distinguished three different scenarios" -this referring to previous work -I have seen this work and they define it as adder, size, and folder -it is not labelled the same way in this work.
This appears to be a misunderstanding.In our previous work (Stojanovski et al. (2022) 14 ), we explored the same models as in this current study: adder, coupled folder, and uncoupled folder.In Stojanovski et al (2022) 14 , we additionally analyzed a combined model for body volume, where L1 was an adder, and L2-L4 was a folder.This analysis, however, is not relevant in the case of the pharynx.Instead, we simulate the pharynx as the three types of models either starting from M1 (shown in main figure) or starting from hatch (in supplemental figures).The reason for starting simulations for the pharynx at M1 in the main text is to exclude confounding impact of higher measurement noise, or other L1-specific effects, for pharyngeal volumes at hatch.
Line 122 -There's a huge jump going from description of simulations which point to an adder mechanism for the pharynx, to talking about the coupling between the organ and the whole body.How does the simulation account for this coupling?Some explanation is lacking here.
We now clarify that the analysis of folder vs. adder does not consider specific mechanisms, such as coupling, and is purely phenomenological (as is also the case for other experimental systems where adders have been observed).The simulations shown in Figure 1  The adder-like behavior of the pharynx suggests that growth is controlled in a size-dependent manner to ensure pharyngeal size uniformity among individuals but does not inform on the mechanism underlying this control.At least two dis nct, not mutually exclusive, classes of mechanisms are conceivable.First, the narrow pharyngeal volume distribu on could be due to precise, ssueautonomous control.Second, pharyngeal volume scaling could involve crosstalk with other ssues.To dis nguish between these two scenarios, we developed an experimental approach perturb pharynx growth and other ssues by ssue-specific auxin-induced degrada on (AID) 15 of the mTORC1 ac vator RagA/RAGA-1 [16][17][18] (Fig. 3a).
Line 182 -lines are referred as epidermal and hypodermal interchangeably in the rest of the paper -please rectify for consistency.
Indeed, we mistakenly used "hypodermal" in the figures and "epidermal" in the text, which is now corrected.The revised text introduces the term epidermal = hypodermal + seam cells once in line 204, and subsequently uses the term epidermal to describe the cells where the col-10 promoter is expressed.
Line 193 -Please mention the actual value of the slope (m) -this would give an idea of the actual scaling.
The values of the slopes are now added to the text (ln.229ff): Without RAGA-1 AID, the slope  of the P-line was 0.44.Under pharyngeal RAGA-1 AID, the P-line was shi ed down by 5-8% but did not systema cally change in slope (Fig. 3d), except for the highest IAA concentra on at the last molt (m = 0.43, 0.41, 0.43, 0.43, 0.37 for IAA from 0 to 1000 μM).Similarly, epidermal RAGA-1 AID caused a near parallel upshi of the P-line by less than 5% without systema cally changing its slope systema cally (m = 0.43, 0.45, 0.45, 0.45, 0.43).
Line 201 -I did not understand the basis for this statement -how do you know the length extension is independent of biomass?
We agree and revised the text accordingly (ln.225): This rapid growth could be due to rapid biosynthesis immediately a er hatching, due to an expansion unrelated to biosynthesis, or due to technical effects and measurement noise.
Line 215 -"We note that while pharynx-to-body proportions were retained (Fig. 3d, e), pharyngeal depletion of RAGA-1 reduced the overall size (Fig. 3c), indicating that total body size and pharynxto-body proportions are controlled by separable mechanisms."Again, the basis of this statement is not clear from the previous section.Please clarify the writing.
Agreed and changed to (ln.245): We note that while pharynx-to-body propor ons were retained (Fig. 3d, e), pharyngeal deple on of RAGA-1 reduced the overall length (Fig. 3c), indica ng that experimental interference can change body length without changing pharynx-to-body length scaling.
Line 303 -"In conclusion, yap-1 is dispensable for growth and development of C. elegans per se, but its activity is crucial for the robustness of pharynx-to-body size proportions under growth imbalance between tissues."Many assumptions are being made here.Does one know that yap-1 doesn't have some sort of pleotropic effect in later larval stages?
Agreed and changed to (ln.339): In conclusion, muta on and knock-down of yap-1 does not reduce the rate of organismal growth and the speed of larval development per se.However, yap-1 is crucial for rapid growth and development, and for normal pharynx-to-body length propor ons, when RAGA-1 is reduced in the epidermis.
Pleiotropic effects in later larval stages are now mentioned in the discussion (ln.374): Consistently, we find that muta on and knock-down of yap-1 alone does not reduce the larval body growth rate of C. elegans (Fig. 5i, j), although it does play an important role in cell polarity 19,20 and aging 21,22 at later stages.
Comments on figures: We are measuring pharynx size immediately after hatching, not in the egg (the first time point of larval development).As shown in the new Supplemental Figure S2, our measurements of pharynx volume and length are precise even in these small animals (~5% error) and precision further improves at later stages (~2%).
We now extensively discuss the implications of measurement noise on our conclusions.Overall, the impact of the measurement noise is very small.See also our answer to reviewer #2.This manuscript combines highly quantitative experiments with modelling to reveal a ultra-sensitive coupling between organ size and growth rate of the body to maintain body proportions.The work is thoughtful, detailed, and provides an important advance.
We thank the reviewer for the endorsement of the quality and impact of our work.
Major issues:

Noise Estimates and Instrument Noise
There are several aspects in the quantification that require clarification in the methods.To show that biological noise estimates are accurate, it is important to show that the noise from non-biological sources are negligible or do not impact the conclusions.Since these issues are technical, the authors should address the questions regarding the set of related points below in the methods.
We now provide quantifications of the noise from non-biological sources and show that this noise does not impact our conclusions (see detailed answers below).
a. What is the pixel size in microns?This parameter limits the accuracy of the size measurements.Does it contribute disproportionately to the CV of length and volume measurements greater in smaller animals?
We now state the pixel size in the methods (ln.418): All experiments were performed on a Nikon Ti2 epifluorescence microscope using a 10x objec ve with NA=0.45 and a Hamamatsu ORCA Flash 4 sCMOS camera with a pixel size of 6.5 μm, leading to an effec ve pixel size of 650 nm in the sample plane.
Note that the pixel size is close to the diffraction limit according to Abbe's definition: r = 2*lamda/(2 NA) = 571 nm.
Below, we provide estimations of the volume error due to the pixel size.In these simulations, we added random noise of -0.5 to 0.5 pixels to the segmented masks after straightening and prior to volume inference to take into account that worm widths are rounded to integer pixel values.This analysis shows that error due to the pixel size is small (maximum 0.6% for pharynx and 0.3% for body volume) and scales according to a power law with the volume.
We do not include this analysis in the revised manuscript, as the pixel size is only one source of noise and is included in our new measurements of the net instrument noise (see answer to point 1b below).
b. What is the net instrument noise due to pixel size, autofocusing, segmentation, and computationally straightening worms?What is their net contribution to the CV?
We now add additional experiments and analyses shown in Supplemental Figures 1c-f, 2, and 3c-d.that address this important point.
Using three independent approaches (see details as provided in the revised methods below), we estimate the net instrument noise for the body volume to be between 0.7% and 1.2%, depending on the larval stage.The net instrument noise for the pharynx volume is 4.8% (hatch), 1.9% (M1), 1.6% (M2), 1.6% (M3), and 1.3% (M4).Instrument noise for length is very similar to that of the volume.
This new analysis validates our speculation from our initial submission that instrument noise for pharyngeal volumes at hatch is higher than at other stages, which allows us to make more clear statements regarding this point in the revised text.Importantly, measurement noise does not impact our conclusions, and has a near negligible contribution to the observed heterogeneity for developmental stages from M1 onwards.
Specifically, we validate the following main conclusions: (i) the biological heterogeneity of the pharynx is smaller than that of the body, also when subtracting noise from non-biological sources.
We correct the observed CV for a contribution instrument noise shown in the revised Figure 1c and in the new Supplemental Figures 1c-f.We can approximate the biological heterogeneity CVbiol as the square root of (CVobserved^2 -CVtechnical^2) (since the variance of the sum of two normal distributions is the sum of the variances of the individual distributions).Figure 1c shows that the CV of pharynx volumes is higher than the CV of body volumes, also after correcting for technical error.Supplemental Figures 1c-f now show that the impact of the technical error is very small, except for volumes at hatch: (ii) the pharynx follows adder-like growth dynamics.
In the new Supplemental Figure 3c-d, we show that the technical noise does not explain the observed adder-like behavior and that the pharynx is indeed distinct from a pure folder.Specifically, we made the following two new analyses: a) Simulations of folder model with technical noise.We ran simulations of a folder model for the pharynx, considering the effect of the technical noise.In the simulations, adding technical noise reduces the slope between ΔV and V1 for a folder to be slightly less than 1.However, for none out of 10'000 iterations of the simulation, did the folder simulations result in slopes as small as what we experimentally observed.We conclude that the pharynx has stronger size control than a folder and that the adder-like behavior is not due to instrument noise.
b) The apparent reduction in the correlation and slope between two variables x and y due to measurement noise is called attenuation bias and can be corrected for if the measurement noise in y is known and independent of x.In the new Supplemental Figure 4c We believe, these additional analyses of technical noise strengthen our manuscript and the conclusions drawn.
Detailed explana ons of these analyses are provided in the revised methods (ln.445 ff) and in Supplemental Figure 2:

Es ma on of net instrument noise
Three independent approaches were taken to es mate the net instrument noise of pharynx and body volume and length.First, standard error of the regression to 10 neighboring point used to es mate volumes at hatch and molts revealed a precision of measurement for hatch to M4 for the pharynx of 4.8%, 1.9%, 1.6%, 1.6%, 1.3% band for the body of 1.2%, 0.96%, 0.82%, 0.70%, 0.87%.Second, volume trajectories were determined for 60 individuals at 5 minutes me resolu on and split into two complementary sets, of which each had 10 minutes me resolu on.The mean difference between the two measurement was close to the standard error of the linear regression.Third, an experiment was conducted, in which for 14 animals were imaged 20 mes every 30 minutes.For each of the 20 images a separate autofocus was performed.The measurement error was determined as the median CV among repeated measurements at each me point.

Determina on of trendlines in of ΔV vs. V1 and comparison to simula ons with technical noise
Trendlines in sca er plots were computed using robust linear regression using the robus it() method of Matlab (v2021b) and default parameters.
To determine the impact of instrument noise, the CV of the biological heterogeneity was es mated by var(biological) = var(total) -var(technical).Star ng volumes V1, biol were then drawn from a normal distribu on with standard devia on corresponding to the biological heterogeneity and sample size corresponding to the number of individuals measured experimentally for the respec ve larval stage.
A folder was simulated by mul plying with a fold-change FC biol drawn from a normal distribu on with standard devia on corresponding to the observed heterogeneity to yield a volume distribu on V2, biol.
Subsequently, technical error was added to V1,biol and V2,biol, drawn from a normal distribu on with standard devia on of the technical noise determined for the respec ve larval stage, yielding simulated V1,biol + tech and V2,biol + tech, from which ΔVbiol + tech = V2,biol + tech -V1,biol + tech was computed.V1,biol + tech and ΔVbiol + tech were normalized to their respec ve means, equivalent to the treatment of experimental data in Figure 3, and the slope of this rela on was determined by linear regression.This simula on was conducted 10'000 mes to compare simula ons to the experimentally observed rela on between with ΔV and V1.

Correc on of regression slopes for a enua on bias
The slope of a linear regression between two observed variables V1, observed and V2, observed is biased towards zero due to measurement error in V1.To correct for this bias (called least squares a enua on bias), we computed the reliability ra o λ , , , , where   , is the variance of V1, corrected for the contribu on of technical error:   ,   ,   , .  , is the experimentally determined measurement error.The measured slopes between V1 and V2 were for then corrected for the a enua on bias by: slopecorrected = slopemeasured / λ.We answer these questions separately below.
Length vs. volume: The reviewer may have misread the CVs shown in the figures.Fig. 1d and Supplemental Fig. 1c show that, at all larval stages, the difference in CV between pharynx volume and length is 2-fold or less.We now show this data more clearly in the new Supplemental Figure 1cf (see above).We note that the technical error of our length measurements is very similar to that of our volume measurements.However, since the biological heterogeneity of lengths is smaller than that of volumes, the technical error has a larger impact for length than for volume.
The volume is inferred by computing the volume of cylindrical slices of 1 pixel width.The length is determined as the total number of these slices, i.e. the length of the midline of the segmented worm.Technical variation in detecting the end points of the worm, has a larger impact on the length than on the volume since the thin tail of the worm adds very little to the volume.On the other hand, volume measurements are affected by noise in width measurements.Together, this may explain why the net instrument noise is very similar for length and volume.
Optical sections: Estimating volumes of shallow objects from optical sectioning is inherently challenging due to the poor axial resolution of fluorescence microscopy.Therefore, the rotational symmetry-based approach that we use, is broadly applied and accepted in the field for C. elegans 23,24 , as well as other rotationally symmetric systems (e.g., bacteria 25 , fission yeast 26 ), which we believe is a superior method to volumetric measurements by optical sectioning.
Below we provide validation of body volume measurements for L4 stage animals, where the limitation of axial resolution is least pronounced.We find good correlation between the planar measurements and optical sectioning.Since the measurements from optical sectioning are likely less precise than those from planar measurements, we feel these data would not significantly add to the manuscript.More importantly, our conclusions using volume estimates and length estimates are highly consistent, suggesting that we are not misled by technical aspects of size estimation.
While volume estimates are useful, the same points can be made with length data in Fig 1 with fewer caveats.Standardising on length would improve consistency across the entire manuscript.
We believe that volume is the appropriate measure for the first part of the manuscript (Figures 1  and 2) for the following reasons: In these figures, we compare the observed heterogeneity in volume to a null model of exponential volume growth.We feel it would not be justified to infer a growth model based on length alone, as the heterogeneity would also be impacted by fluctuations in the width-to-length aspect ratio, e.g., due to changes in worm posture.Moreover, as we show in the new supplemental Figures 2c and e, estimations of heterogeneities in length are more sensitive to technical noise.This is because the biological variability in length is smaller while the technical noise is similar for length and for volume.
The second section of the paper does not consider individual animals, but instead how the population mean varies across different conditions.This allows us to average over many individuals, such that individual-to-individual differences in the width-to-length aspect ratio do not impact our conclusions.Importantly, however, our conclusions also hold for volume-based analysis as shown in the supplemental figures.

Role of yap-1
There are some caveats to the analysis of yap-1 and potentially other models for yap-1's role.
a.The role of yap-1 is depicted as a major point in the manuscript based on the prominence in the title and abstract.However, the evidence for the role of yap-1 is solely based on RNAi, which may not be specific.It is important to validate this result with an outcrossed yap-1 mutant.Alternatively, the authors can be explicit about the caveats and reduce the prominence of yap-1 in the manuscript.
We now include additional experiments using a previously characterized and extensively outcrossed allele yap-1(tm1416) that deletes the WW domain of YAP-1.This mutant allele recapitulates the sensitivity of yap-1(RNAi) regarding deviations in pharynx size upon epidermal AID of RAGA-1.The mutant also shows good agreement with the RNAi for other phenotypes: growth rate, developmental speed, and pharyngeal length at L4 to adult transition.
Overall, the phenotype of the yap-1(tm1416) allele is slightly weaker than that of the RNAi.Most strikingly, the mutant allele does not cause larval arrest when combined with epidermal AID of RAGA-1.These data suggest that tm1416 is not a complete null allele.Alternatively, larval arrest could be due to unspecific RNAi.We discuss these two possibilities in the revised manuscript.
The yap-1(tm1416) mutation enhances larval arrest of animals treated with yap-1 RNAi (and epidermal RAGA-1 AID).This shows that the yap-1(tm1416) can cause larval arrest, albeit this function is only apparent when combined with yap-1(RNAi).These data make us favor the interpretation that tm1416 is not a complete null allele, rather than arrest being due to unspecific RNAi.Final evaluation of this point will require generation and detailed characterization of a full deletion allele by CRISPR/Cas 9 in future work.
Together, these results strengthen our evidence that yap-1 is important for maintaining pharynx-tobody proportions as stated in the revised title and abstract.b.To justify the title that yap-1 mediates ultra-sensitive coupling would require additional experiments to show that the Hill co-efficient is reduced when yap-1 activity is disrupted.Alternatively, this point can be addressed by revising the title to downplay this specific claim, which would in no way reduce the importance and interest of this work.
We agree with the reviewer and have adjusted the title accordingly, which now reads: Maintenance of appropriate size scaling of the C. elegans pharynx by YAP-1 c. yap-1 signalling is presented as a general mechanism for robustness of proportionate growth.Is it not just as likely that yap-1 specific to the raga-1 pathway as there is literature relating mTOR signalling to Hippo/YAP (e.g., Csibi & Blenis, Nature Cell Biology 2012)?These alternative models should be mentioned in the discussion.This is indeed a possibility, which we now discuss in ln.380 of the revised manuscript: In future research, it will be important to address if YAP-1 globally senses growth or size imbalances, or if its response is mediated by direct crosstalk between the mTOR and the Hippo pathway 27 .
The reference cited is the primary ar cle discussed by in the News & Views ar cle of Csibi & Blenis.
Minor issues: Does raga-1 affect RNAi efficacy?The authors suggest that yap-1 only comes into play when there is raga-1 perturbation.But there is no evidence provided to rule out the possibility that the RNAi is more severe under raga-1 loss.The point that other RNAi treatments do not synergize with raga-1 does not address this issue since those RNAi treatments may be true negatives.This caveat should be stated in the results section.
We now confirm the RNAi phenotypes using the genetic allele yap-1(tm1416) instead of RNAi (Figure 5).Since we do not use RNAi in these experiments, we can exclude that the phenotypes of epidermal RAGA-1 AID strains is due to potential differences in RNAi efficiency.
Introducing sizing mechanisms (e.g., adder, folder) in greater detail would provide context to make it easier for readers to understand and appreciate many aspects of this work.
Adders and sizers are now introduced in greater detail in the second paragraph of the introduction (ln.32 ff.): At the scale of individual cells, size homeostasis has been extensively studied by time-lapse microscopy of yeasts, bacteria, and mammalian cells.In cells, stochas c size fluctua ons are corrected within a few cell divisions.For many cell types, larger cells on average undergo a smaller volume fold change per cell cycle than smaller cells.Thereby, cells that deviate from the norm return to a stable reference point.Depending on how fast this reference point is reached, cells are called to follow adder or sizer mechanisms 25,[28][29][30][31] .A sizer refers to cell types that, on average, return to the appropriate size within one cell cycle such that their size at division is independent of their size at birth.An adder refers to cells that, on average, grow by a constant absolute volume, independent of their size at birth.Unlike sizers, adders take mul ple cell cycles to return to a reference point.

Fig 1 a
Fig 1 aAre your really measuring egg pharynx size?Can authors speak to the resolution they can obtain on their platform with really small pharynx.Mentioned briefly in line 108 -but not enough is said.

Fig 3 d .Reviewer # 2 (
Fig 3 d.Can the legend be improved -what are circles, squared etc? colour code is fine as it is consistent across -representing IAA concentration.

Fig 2
Fig 2 could benefit from a color scale bar for a-b and c.

Fig
Fig 3e -the sizes of the circles are obscured by the error bars.A larger panel could address this visualization issue.
µabs stands for (dV/dt), µ stands for (dV/dt)/V = d log(V)/dt This is now specified more clearly in the revised text in ln.98ff.Throughout this ar cle, growth rate refers to the change in log transformed volume per me (µ = d log(vol) / dt = (d vol/dt)/vol), i.e., the growth rate normalized to the current size, unless specified as the absolute growth rate (µabs = d vol / dt), which indicates the absolute change in volume per me.
do therefore indeed not account for the coupling.Importantly the work shown in Figures3,4and 5 goes beyond the phenomenological description of an adder and presents mechanistic insight in how pharyngeal size scaling is achieved.The corresponding section now reads (175ff):

Fig 1 a:
Fig 1 a: Are your really measuring egg pharynx size?Can authors speak to the resolution they can obtain on their platform with really small pharynx.Mentioned briefly in line 108 -but not enough is said.

Fig 3 d
Fig 3 d.Can the legend be improved -what are circles, squared etc? colour code is fine as it is consistent across -representing IAA concentration.The visualization is now improved.The apparent squares in the previous figure were due to the cap of the error bars.We now show error bars in black and without cap, such that circles are visible more clearly.Where invisible, the error bar is smaller than the marker.
, we show that the corrected slopes remain smaller than what is expected for a folder model and are close to an adder model.Note that, in Supplemental FigureS4, we plot the relation between V1 and V2 (not between V1 and ΔV like in the main figure).We do this in order to meet the requirement of independence of the variables for computing the attenuation bias.In this case the expected slope for an adder model is non-zero and indicated in blue in the graphs.The precise slope expected for an adder depends on the mean volume fold change undergone in a larval stage as follows: slope = 1/(fold change).[Detailsregarding this relation were discussed in our previous work(Stojanovski et al., 2022) 14 ].
c. How are the errors propagated in the estimation of volume and does that account for the approximately 10-fold difference in the observed CV of pharynx length vs volume in Fig 1d vs Supplemental Fig 1c?Is there validation by comparing the estimates from taking multiple optical sections?
The figure shows volumes estimated from optical sectioning plotted against volumes estimated from planar measurements.Each circle is a different time point of a micro chamber experiment imaged every 10 minutes.Colors (yellow, blue, red) correspond to different individuals.This experiment was conducted on a spinning-disc confocal microscope (instead of a wide-field microscope) and at double the magnification of what we used in the manuscript (20x, 0.75 NA instead of 10x, 0.45 NA).

Fig 2
Fig 2 could benefit from a color scale bar for a-b and c.A color scale bar has been added.Fig 3e -the sizes of the circles are obscured by the error bars.A larger panel could address this visualization issue.We improved this visualization by showing the error bars in black and without cap.Instead of circle size, we use different marker types to indicate the IAA concentration.Typo in Fig 2 legend: "...produces and adder-like..." DONE.Typo in Supplemental Fig 2c-d y-axis label: "voulme" DONE.